Rifampicin‐resistant RpoB S522L Vibrio vulnificus exhibits disturbed stress response and hypervirulence traits

Abstract Rifampicin resistance, which is genetically linked to mutations in the RNA polymerase β‐subunit gene rpoB, has a global impact on bacterial transcription and cell physiology. Previously, we identified a substitution of serine 522 in RpoB (i.e., RpoBS522L) conferring rifampicin resistance to Vibrio vulnificus, a human food‐borne and wound‐infecting pathogen associated with a high mortality rate. Transcriptional and physiological analysis of V. vulnificus expressing RpoBS522L showed increased basal transcription of stress‐related genes and global virulence regulators. Phenotypically these transcriptional changes manifest as disturbed osmo‐stress responses and toxin‐associated hypervirulence as shown by reduced hypoosmotic‐stress resistance and enhanced cytotoxicity of the RpoBS522L strain. These results suggest that RpoB‐linked rifampicin resistance has a significant impact on V. vulnificus survival in the environment and during infection.


| INTRODUCTION
Antibiotic resistance is of growing concern and poses a severe threat to human health and the global economy (WHO, 2014).Over the past decades, efforts have been increasingly made to understand the occurrence and dissemination of antibiotic resistance (Naylor et al., 2018;Zheng et al., 2021) and to find alternatives to classical antimicrobial treatments (Ghosh et al., 2019;Kumar et al., 2021).
A consequence of antibiotic resistance is the pleiotropic effect that mutations conferring resistance can have on bacterial physiology and, consequently, the dissemination and evolution of antibiotic resistance (Gifford et al., 2018;Maharjan & Ferenci, 2017;Schenk & de Visser, 2013).In this work, attention is directed to the effects of a mutation conferring rifampicin resistance on stress responses and virulence in the human pathogen Vibrio vulnificus.V. vulnificus is a gram-negative marine bacterium that causes severe gastroenteritis or septicemia through consumption of undercooked seafood, in particular oysters, or through wound infections via contact with contaminated seawater (Oliver 2005(Oliver , 2015)).It exhibits features of an opportunistic pathogen as successful infection is favored by preexisting health conditions, such as hemochromatosis (Bullen, 1991;Linkous & Oliver, 1999;Wright et al., 1981).Infection is fatal in approximately 50% of patients, as a result of septicemia and organ damage (Jones & Oliver, 2009).
Like most human pathogens, survival in the environment and the human host are key stages of the V. vulnificus infection process (Fang et al., 2016) along with the production of a range of toxins and exoenzymes (Miyoshi, 2013;Oliver et al., 1986;Pérez-Reytor et al., 2018).The influence of mutations conferring rifampicin resistance on physiological fitness and virulence during infection has so far received little study for V. vulnificus (Cutugno et al., 2020).
Antibiotic resistance, such as resistance to rifampicin, is often reported in both clinical and environmental isolates of V. vulnificus (Ottaviani et al., 2001) and other Vibrio spp.(Heng et al., 2017;Oh et al., 2011).Phenotypic characterization of rifampicin-resistant V.
vulnificus would enable prediction of their ability to thrive and establish themselves in the environment and to cause infections in humans.
In the present work, the focus was on two spontaneous rifampicin-resistant V. vulnificus strains carrying a serine-to-leucine amino acid exchange at residue 522 in RpoB (RpoB S522L ).This amino acid exchange is of particular significance because it has previously been shown to negatively impact the growth of V. vulnificus, while wild-type levels of tolerance to ethanol, hyper-osmotic, and acidic stress conditions were maintained (Cutugno et al., 2020).Furthermore, substitutions in the same amino acid in Escherichia coli RpoB are associated with a stringent-like phenotype (Alifano et al., 2015;Murphy & Cashel, 2003).
In this study, we show that transcription of specific sets of stress and virulence genes is significantly altered in V. vulnificus expressing RpoB S522L , thereby leading to changes in stress survival and cytotoxicity, and suggest that RpoB-linked rifampicin resistance impacts the ability of the organism to thrive in the environment and to infect humans.

| In silico evaluation of the S522L mutation
To evaluate the potential structural consequences of the S522L amino acid substitution in V. vulnificus RpoB we interpreted the E. coli RNA polymerase holoenzyme structures (PDB-ID: 7KHB) with bound ppGpp (PDB-ID:7KHI).Structures were downloaded from the PDB database (rcsb.org)and visualized and evaluated in Pymol (https:// pymol.org/2/).

| Growth characterization
Growth was assessed at 30°C in LBN or CDM and at 37°C in Dulbecco's modified eagle medium (DMEM).For the growth characterization in CDM and LBN, the strains were grown overnight in 20 mL LBN at 30°C.In initial experiments, overnight cultures were serially diluted and plated on LBN agar, and following overnight incubation the number of colony-forming units was counted to verify equitable numbers of viable bacteria in the culture of each strain.
Bacterial cultures were then washed and diluted in 30 mL LBN or 30 mL CDM to a final OD 600 of 0.05 in 250 mL flasks.The flasks were incubated at 30°C with 150 rpm agitation.OD 600 was measured every 60 min for the first 10 h and lastly at 24 h.The specific growth rate (μ) between two time points (t1 and t2) taken during the exponential phase was calculated using the formula μ = ln2/g, where g is the doubling time.At least three biological replicates were used for each strain.To study growth in DMEM, the strains were analyzed in microtiter plates.Overnight cultures were grown in 2 mL LBN at 37°C and, after a washing step, diluted in DMEM at an initial OD 600 of 0.01.Three biological replicates were used for each strain and each of them was assessed in two technical replicates.The plates were incubated statically in an LT-5000 MS microtiter plate reader at 37°C and the OD 600 was measured, following 5 s of shaking, every 15 min for 48 h.
To assess the minimum required concentration (MRC) of NaCl, a microtiter plate was set up with two-fold dilutions of NaCl in Mueller-Hinton (MH) broth, from 0.4 to 0.03 M, and broth without additional salts was used as a blank.MH (peptone 17.5 g/L, meat extract 2 g/L, starch 1.5 g/L) was selected as a growth medium in which V. vulnificus CMCP6 would grow without NaCl supplementation.
The osmolality of MH broth is 0.3 Osm/kg.CMCP6 failed to grow on tryptone 10 g/L, yeast extract 5 g/L (0.1 Osm/kg).All the strains were cultured overnight in LBN at 37°C and, after a washing step, inoculated in the microtitre plate to an initial OD 600 of 5 × 10 −4 .Three biological replicates were used for each strain and each of them was assessed in two technical replicates.The plates were incubated for 24 h at 30°C and the final OD 595 was measured with a Sunrise microtiter plate reader.The viability of the bacteria at the end of the experiment was assessed by determining the number of cfu/mL.The bacteria were serially diluted in 10-fold steps and 10 µL was spotted in triplicate onto LBN agar.After 24 h incubation at 37°C the number of colonies was counted and the cfu/mL calculated.
To test the growth on solid media in the absence of NaCl, the Briefly, the strains were grown overnight on LBN agar at 37°C.Several single colonies were selected for resuspension in 0.85% NaCl to reach OD 600 0.1.A sterile cotton swab was dipped into the cell suspension and streaked on MH agar.MH + 0.4 M NaCl agar was used as a control.Plates were allowed to dry at room temperature and incubated at 37°C for 16 h.

| RNA extraction and qRT-PCR gene transcription analysis
Once the phenotypic traits common to both Rif R 8 and Rif R 9 were determined, the associated transcriptional analysis was performed solely on the Rif R 9 strain.For RNA extraction, cell pellets were harvested from overnight cultures in 2 mL LBN at 37°C or exponential (OD 600 0.5) and stationary (OD 600 2.5) phase during growth in 30 mL LBN at 30°C.Two volumes of RNAprotect (Qiagen) were mixed with one volume of bacterial culture containing approximately 0.5 OD 600 of cells and processed following the manufacturer's instructions.To lyse the bacterial cells, the pellet was re-suspended in 200 µL TE buffer (30 mM Tris-HCl, 1 mM EDTA, pH 8.0),15 mg/mL lysozyme,1 mg/mL Proteinase K and incubated at room temperature for 10 min.
The lysate was then used for RNA extraction with an RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol.RNA was eluted in 50 µL RNase-free water and a rigorous DNase treatment was performed using the TURBO DNA-free kit (Life Technologies), according to the manufacturer's instructions.RNA integrity was verified on a 1.5% agarose gel and concentration and quality were assessed using a NanoDrop spectrophotometer.Only the samples with a 260/280 ratio ≥1.9 were used for the qRT-PCR protocol.
The Transcriptor First Strand cDNA Synthesis Kit (Roche) and the Random Hexamer Primer provided by the manufacturer, were used to synthesize cDNA from 1 µg total RNA.The RT reaction was carried out as follows: 10 min at 25°C followed by 60 min at 50°C.Primer-BLAST (Ye et al., 2012) was used to design the primers for the qPCR reactions (Table 1) and, before use, the primer specificity and efficiency were calculated.Two microliters of a 1:10 dilution of the cDNA were used in a 10 µL reaction to study transcription levels of the several gene targets through qPCR, using the SYBR Green I Master (Roche) and the LightCycler 480 (Roche).Two technical replicates were utilized to test each reaction.The transcription of each gene was normalized using tuf (elongation factor Tu) as endogenous control, similar to previous transcription studies (Nowakowska & Oliver, 2013).Fold changes in transcription levels were calculated using the Pfaffl method (Pfaffl, 2001).

| Cytotoxicity assay
To study cytotoxicity, HeLa cell lysis was assessed following coincubation with bacterial cells or bacterial culture supernatant.In both cases, HeLa cells were seeded in 48-well plates with DMEM + 10% fetal bovine serum (FBS) + 1% pen/strep.Before co-incubation, the HeLa cells were washed twice with DMEM + 10% FBS without antibiotics and allowed to recover for 30 min before adding the bacterial cells or the bacterial supernatant.Bacterial cultures were grown overnight in LBN broth at 37°C.To test the cytotoxicity of the supernatant, 1 mL overnight culture was centrifuged at 13,000 rpm for 2 min and the supernatant was filtered using a 0.22 µM filter.One hundred microliters filtered supernatant was added to the well containing the HeLa cells in 900 µL DMEM.For the co-incubation T A B L E 1 Primers for quantitative real-time PCR (qRT-PCR) of stress and virulence target genes in Vibrio vulnificus.coli, displaying 85% sequence identity and 92% conservation at the amino acid level.
In the E. coli structure, serine 522 forms a hydrogen bond with aspartate 516 of RpoB, and this bond appears to be critical for the productive action of rifampicin at the RpoB-RNAP complex.It has been shown that the substitution of aspartate 516 confers high levels of rifampicin resistance and it is frequently detected in clinical isolates of rifampicin-resistant bacteria (Andre et al., 2017).The serine 522 to leucine substitution of RpoB would disrupt the hydrogen bond between serine 522 and aspartate 516.Therefore, we speculate that the serine-to-leucine substitution at residue 522 has the potential to alter the geometry of the active site of RpoB, and thus to change the transcriptional activity of RNAP (Figure 1).
Interestingly, in Streptomyces coelicolor rifampicin resistance mutations located in rifampicin cluster I of rpoB restore the ppGppdependent production of the antibiotic actinorhodin in relA and rplK strains that fail to induce a stringent response (Xu et al., 2002).
Among the amino acid substitutions identified in the S. coelicolor 3.2 | The S522L substitution causes a growth defect in rich media, but not in chemically defined media We had previously demonstrated that two Rif R V. vulnificus strains (Rif R 8 and Rif R 9), carrying the S522L amino acid substitution in RpoB, have a growth defect in LBN medium (Cutugno et al., 2020).In this work, the wild-type CMCP6 and the RpoB S522L V. vulnificus strains were assessed for growth in both nutrient-rich and nutrient-limited conditions.To this end, the wild type and Rif R 8 and Rif R 9 were grown at 30°C in 30 mL LBN or CDM (Figure 2).The rifampicin-resistant strains showed a growth defect in LBN medium compared to the wild type, with a slower growth rate during exponential growth, though eventually achieving the same biomass accumulation in stationary phase.This difference was not observed in CDM (Figure 2).A similar growth phenotype has been described for other bacterial species with mutations in RNAP that cause a stringent-like phenotype, mimicking the presence of (p)ppGpp (Conrad et al., 2010;Murphy & Cashel, 2003).
To investigate whether RpoB S522L V. vulnificus influenced the stringent response, we investigated the transcription of rmf, encoding ribosome modulation factor, and fusA, encoding translation elongation factor G (EF-G), which are up-and downregulated by (p) ppGpp in E. coli, respectively (Izutsu et al., 2001;Zhang et al., 2016).
Gene transcription analysis of wild-type V. vulnificus revealed reduced transcription of the fusA gene in stationary phase compared to exponentially growing bacteria in LBN, as expected due to the general downregulation of the translation machinery upon induction of the stringent response when bacteria enter stationary phase (Pletnev et al., 2015).Conversely, rmf transcription was increased in the stationary phase, in line with previous studies (Yamagishi et al., 1993).While there was no significant difference in fusA transcription between the wild type and the Rif R 9 mutant, there was a 3.5-fold higher rmf transcription during stationary phase in Rif R 9 as compared to CMCP6 (Figure 2c).These data suggested that the RpoB S522L variant may influence the stringent response.

|
The RpoB S522L variant alters the hypoosmotic stress response of V. vulnificus and the transcriptional profile of stress-related genes Coastal and intertidal bacteria, such as Vibrio, must adapt to changing seawater salinities caused by daily tides and weather conditions (Jones et al., 2008;León Robles et al., 2013;Randa et al., 2004).Previous work in E. coli has shown that certain rpoB variants affect osmotic stress responses (Xiao et al., 2017).We recently reported that V. vulnificus strains with RpoB S522L have a similar hyperosmotic stress response to that of the wild type (Cutugno et al., 2020).To further investigate the response of the RpoB S522L strain to osmotic stress, hypoosmotic stress tolerance was analyzed in MH media which allows the growth of Vibrio spp. in the absence of supplementary NaCl (CLSI, 2015).MRC broth experiments revealed that both S522L mutants Rif R 8 and Rif R 9 reached a significantly lower optical density than the wild type after 24 h culture at low concentrations of NaCl (≤50 mM), while NaCl concentration had little influence on OD 600 measurements of the wild type (Figure 3a and Appendix: Figure A1).When growth was assessed on MH agar, the wild type grew equally well with or without NaCl supplementation, while RpoB S522L strains showed a clear dependence on NaCl supplementation (Figure 3b).Similar numbers of viable bacteria (9.7-12.9× 10 8 cfu/mL) were recovered for all three strains following 24 h culture in MH broth supplemented with 0.4 M NaCl (Figure 3c).In MH broth, the cfu/mL values for the 3 strains were much lower (9.3-40.6 × 10 4 cfu/mL) and similar to the concentration of the starting inoculum (5 × 10 5 cfu/mL).The low viability of the wild type was unexpected given its OD 600 value and its strong visible growth on MH agar.
Hyper-and hypo-osmotic stresses pose very different physiological challenges to the bacteria and the two types of response are generally opposing, with compatible solute synthesis and influx pumps used in response to high salt concentrations, while water influx, mechanosensory channels and transient pores are involved in the response to osmotic downshift (Gregory & Boyd, 2021;Rao et al., 2013).To probe if the hypoosmotic stress-sensitive phenotype of the Rif R 9 strain was correlated with a general dysregulation of the osmotic stress response, transcription of putA was analyzed in nonstressed cells during growth in LBN media.putA encodes proline dehydrogenase, which converts proline into glutamate-the major osmolyte required during hyperosmotic stress adaptation of V.
vulnificus (Lee et al., 2003).putA transcription showed a trend, albeit not quite reaching statistical significance, towards higher levels in the Rif R 9 strain than the wild type during exponential growth and in stationary phase (Figure 4).These results suggest that V. vulnificus expressing RpoB S522L is characterized by an increased basal level of stress gene transcription.

| The RpoB S522L variant increases cytotoxicity through increased transcription of vvhA hemolysin
RpoS is a global regulator of stress responses (REF).Additionally, RpoS works together with ToxR to regulate virulence, including toxin and exoenzyme production, through mediating cross-talk between stress response and virulence regulatory pathways (Chowdhury et al., 1996).ToxR is a global virulence regulator that senses cyclic dipeptides and is a component of the signaling network that stabilizes rpoS mRNA (Kim et al., 2018).Transcription of rpoS, toxR, and one of the latter's downstream effectors, ompU, was analyzed.These analyses showed increased transcription of rpoS, toxR, and ompU in the Rif R 9 strain compared to the wild type during exponential growth and stationary phase in LBN (Figure 4).The increased rpoS transcription provides additional evidence for the increased basal level of stress gene transcription associated with V. vulnificus expressing RpoB S522L .
RpoS upregulates the production of both the RTX toxin and the VvpE elastase (Guo et al., 2018;Hülsmann et al., 2003) while ToxR is a positive regulator of VvhA hemolysin production (Lee et al., 2000).
To examine the effects of the S522L substitution on virulence, the cytotoxicity of wild type and RpoB S522L V. vulnificus was examined.
Bacteria were co-incubated with HeLa cells in DMEM and cell lysis was subsequently evaluated through LDH quantification (Matlawska-Wasowska et al., 2010).The Rif R 8 and the Rif R 9 strains displayed delayed cytotoxicity compared to the wild type, as demonstrated by the additional 2-3 h co-incubation time needed to cause maximum eukaryotic cell lysis (Figure 5a).Although there were identical numbers of bacteria in the inoculum (1 × 10 6 cfu/mL), visualization of the co-incubated cells at later time points showed a greater abundance of wild-type bacteria than of RpoB S522L V. vulnificus (Figure 5b).This is particularly evident at the 4 h time point where it is possible to distinguish individual RpoB S522L bacteria, whereas the wild type has increased in density to such an extent that the bacteria appear confluent.The continued viability and replication of both Rif R 8 and Rif R 9 is shown by their increase in bacterial density between 4 and 7 h (Appendix: Figure A2) and by the increase at 5 h in the number of viable bacteria to 2.6 × 10 7 and 5.0 × 10 7 cfu/mL, respectively.In contrast, the wild type reached 3.0 × 10 8 cfu/mL at the same time point.Moreover, in the latter sample, HeLa cells display a greater degree of morphological disruption.To investigate whether differential bacterial growth rates in DMEM eukaryotic cell culture media could be the cause of the delayed cytotoxicity kinetics, the growth of the three strains was analyzed and revealed a major growth defect associated with the RpoB S522L variant in this medium (Figure 5c).
V. vulnificus cytotoxicity is contact-independent (i.e., it does not require direct contact between bacteria and host cells), rather cytotoxic effector proteins are secreted into the extracellular milieu where they bind and enter target cells via surface receptors.This allowed us to examine cytotoxicity in a growth-independent manner, whereby the supernatant of bacterial cultures grown in LBN to stationary phase (16 h growth, OD 600 2.5) was incubated with HeLa cells to evaluate lysis.In this experiment, the cell-free supernatants of the two S522L mutants showed faster and significantly higher cytotoxicity than the wild-type supernatants (Figure 6a).This suggested that during the pre-incubation culture, the RpoB S522L strains had produced and secreted a greater amount of cytotoxic proteins into the culture media.qRT-PCR analysis of these preincubation cultures, which had been grown in LBN to stationary phase, revealed significantly increased transcription of the toxR-dependent vvhA hemolysin gene in the RpoB S522L V. vulnificus compared to the wild type and a nonstatistically significant increase in the transcriptional levels of the rpoS-dependent rtxA toxin gene, while no differences were observed for the vvpE elastase gene (Figure 6b).Gross , 1989;Jin et al., 1988aJin et al., , 1988b;;Wichelhaus et al., 2002).
Evaluation of these effects is key to understanding how antibiotic resistance, already challenging per se, could affect the chances of a resistant microorganism surviving and spreading both in the environment and during infection (Gifford et al., 2018;Schenk & de Visser, 2013).Moreover, previously reported effects of rifampicin resistance on virulence traits of human pathogenic bacteria, show that such mutations can affect the success of the infection process (Björkman et al., 1998;Gao, et al., 2012).For these reasons, we investigated at phenotypic and transcriptional levels the effects of the S522L rpoB mutation in V. vulnificus.
We first analyzed the position of the RpoB residue S522 in the structural model of the E. coli RNAP, which has a high level of sequence conservation with the V. vulnificus enzyme.We observed that, although not close to the (p)ppGpp binding site, S522 is located in the active site region of the RNAP where mutations have the potential to alter the geometry of the site with possibly dramatic consequences for transcription.We demonstrated that the mutant strain growth defect is unique to growth in rich LBN media, while the growth rate is equal to or higher than the wild type in CDM.
Mutants with altered transcriptional machinery can exhibit a range of phenotypes (Alifano et al., 2015).In this work, we focused on the effects of the RpoB S522L variant on the stress response, to provide data for predicting the potential survival and spread of the mutation in the population.Our previous report showed that the S522L mutants had a similar tolerance for high NaCl concentrations as the wild type (Cutugno et al., 2020).In contrast, here we showed a sensitivity of RpoB S522L V.
vulnificus to hypoosmotic stress and their inability to grow on MH media without NaCl supplementation.During these experiments there was visible growth of the wild type on both MH broth and agar, however, its viability was low after 24 h culture in MH broth.We hypothesize that the wild-type bacteria underwent an initial period of replication, but that continued incubation in a hypoosmotic solution resulted in a reduction of cfu.In contrast, RpoB S522L variants were unable to replicate in the hypoosmotic stress environment.As a possible cause of this unique stress survival profile, we observed increased transcription in the RpoB S522L variants of the hyperosmotic stress-related gene putA (Lee et al., 2003) and two major global regulators in Vibrio spp., rpoS and toxR.
RpoS and ToxR control the expression of large regulons, with effects on growth, stress response, and virulence in many Gram-negative human pathogens (Battesti et al., 2011;Dong & Schellhorn, 2010; Clinical and Laboratory Standards Institute (CLSI) M45 protocol was applied (Clinical and Laboratory Standards Institute [CLSI], 2015).

|
The S522L substitution maps to a region in RpoB critical for RNA polymerase function Serine 522 is located in rifampicin cluster I of the RNA polymerase β-subunit(Alifano et al., 2015;Cutugno et al., 2020) where the majority of amino acid substitutions conferring rifampicin resistance occur(Alifano et al., 2015) but it is not directly involved in binding rifampicin(Alifano et al., 2015).First, we wanted to understand the structural consequences of the exchange of serine 522 to leucine on RpoB in the context of the RNA polymerase holoenzyme (RNAP).For this purpose, we employed a highresolution structure of E. coli EcRpoB bound within EcRNAP to infer possible consequences of the serine 522 to leucine substitution in RpoB.VvRpoB is highly conserved with its counterpart in E.
study was ScRpoB S423T , which corresponds to serine 522 in the VvRpoB protein.This observation suggests that the rifampicin resistance conferred by VvRpoB S522L could also functionally mimic the modifications induced in the RNA polymerase by binding of ppGpp(Xu et al., 2002).Although the exact physiological consequences of the RpoB S522L substitution are difficult to predict for V. vulnificus, it seems possible that it could have an impact on gene transcription and growth of V. vulnificus in response to stress conditions, which merited further investigation.F I G U R E 1 The RpoB S522L variant potentially affects geometry of the transcription initiation bubble.The Escherichia coli RNA polymerase rrnBP1 promoter open complex (PDB-ID: 7KHB) is shown on the left.The zoom inset on the right-hand side shows RpoB S522 forming a hydrogen bond with RpoB D516 .Residue D516 is located within the loop region of RpoB that interacts with the DNA bulge in the open complex.
Growth profile of RpoB S522L Vibrio vulnificus.Growth curves of V. vulnificus CMCP6 wild type (WT) (black), Rif R 8 (dashed green), and Rif R 9 (solid green) in (a) LBN and (b) chemically defined medium (CDM).The data points are the mean values of at least five biological replicates.The growth rates of each strain and the ratio between mutant and wild-type growth rates are reported below the graph.Student's t-test compares the growth rate of each RpoB S522L strain to the wild type.(c) rmf and fusA gene transcription in V. vulnificus CMCP6 wild type and Rif R 9 during exponential (EXP) and stationary (STAT) growth in LBN.Normalized gene transcription values, using tuf as the reference, are reported.Values presented are the mean ± SD of three biological replicates.Student's t-test was performed comparing transcription in the wild type and the mutant (*p ≤ 0.05; ns,p > 0.05).
4 | DISCUSSIONSecondary effects of rpoB mutations conferring rifampicin resistance have been characterized in several bacterial species(Alifano et al., 2015;Billington et al., 1999;Cutugno et al., 2020; Jin & F I G U R E 4 Increased stress-related gene transcription in RpoB S522L Vibrio vulnificus.Transcription of four genes (rpoS, putA, toxR, and ompU) in V. vulnificus wild type (black) and Rif R 9 (green) at exponential and stationary growth phase in LBN, in the absence of stress, was analyzed.Normalized gene transcription values, using tuf as the reference, are reported as the mean ± SD of three biological replicates.Student's t-test compared transcription in the wild type and in the mutant.*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.F I G U R E 5 (See caption on next page).

F
I G U R E 6 Elevated contact-independent cytotoxicity of RpoB S522L Vibrio vulnificus.(a) The cell-free supernatant of overnight cultures of V. vulnificus wild type (black), Rif R 8 (dashed green), and Rif R 9 (solid green) grown in LBN was incubated with HeLa cells.The percentage of lysed HeLa cells, compared to the 100% lysis control, was calculated through LDH quantification each hour between 5 and 9 h after the addition of the supernatant.Reported values are the mean ± SD of three biological replicates.Student's t-test compared the wild type and the variants.*p ≤ 0.05.(b) Toxin and exoenzyme gene transcription in V. vulnificus wild type (black) and Rif R 9 (green) in overnight pre-cultures in LBN.The transcription of vvhA, vvpE, and rtxA was analyzed, and normalized gene transcription values, using tuf as the reference, are reported as the mean ± SD of three biological replicates.Student's t-test compared transcription in the wild type and in the variant.**p ≤ 0.01.F I G U R E 5 Cytotoxicity of wild type and RpoB S522L Vibrio vulnificus in coculture conditions.(a) Coincubation of bacteria with HeLa cells and LDH quantification.The percentage of lysed HeLa cells, compared to a 100% lysis control, was calculated through LDH quantification at each hour after the addition of the wild type and RpoB S522L V. vulnificus inoculum.The plates were not centrifuged, rather the bacteria were allowed to settle onto the cells.Reported values are the mean ± SD of three biological replicates.Student's t-test compared the wild type and the variants.*p ≤ 0.05; **p ≤ 0.01.(b) Light phase microscopic images of coculture samples after 4 and 7 h of coincubation to demonstrate bacterial cell density.Bacterial cells or groups of bacterial cells are indicated by black arrows.HeLa cells are indicated by white arrowheads.(c) Growth curve of V. vulnificus CMCP6 wild type (black), Rif R 8 (dashed green), and Rif R 9 (solid green) in Dulbecco's modified eagle medium (DMEM).Data values are the mean of three biological replicates.

F
I G U R E A1 Complete NaCl minimum required concentration (MRC) profile of Vibrio vulnificus CMCP6 wild-type (black) and S522L mutants (green) in Mueller-Hinton media.Growth at NaCl concentrations between 0.4 and 0 M was assessed in Mueller-Hinton broth.OD595 was measured after 24 h incubation at 30°C.Three biological replicates for each strain were tested and the reported values are the mean of the three replicates.Student's t-test was performed comparing S522L strains with the wild-type and p-values are shown (**<0.01;***<0.001).APPENDIX A F I G U R E A2 Cytotoxicity and viability of RpoBS522L Vibrio vulnificus in coculture conditions.Light phase microscopic images after 4, 5, 6, and 7 h of coincubation of RifR8 and RifR9 bacteria with HeLa cells.